The use of eukaryotic cells, and in particular animal cells, for the expression of cloned heterologous genes is the subject of numerous research studies. Indeed, only eukaryotic cells are capable of carrying out the post-translational modifications which are, in numerous cases, necessary for obtaining an active protein. At present, yeast constitutes one of the most widely used eukaryotic expression systems, because it is easy to use; however, the glycosylation system of yeasts, which differs somewhat from that of animal cells does not permit certain proteins to be obtained in active form.
In order to obtain the expression of a gene in an animal cell, vectors of viral origin are used in particular. Among these, there may be mentioned baculoviruses which are currently used in numerous laboratories. Indeed, these viruses have the following advantages; they permit the insertion of long segments of DNA and possess, in addition, several strong promoters which are active at various stages of the virus replication cycle. These promoters are capable of inducing an extremely high level of expression of the genes placed under their control. Two late promoters, the polyhedrin promoter, and the protein P10 promoter, have been more particularly used for the expression of heterologous genes.
However, these baculovirus-derived expression vectors possess certain disadvantages which limit their use: first of all, the heterologous gene inserted into one of these vectors is expressed only within the framework of a viral infection; now, the viral infection results rapidly in the lysis of the cells, which means that the expression of a gene in the latter can only be transient, and which results, moreover, in the rapid degradation of the proteins produced by the cell. In addition, insofar as the heterologous gene is placed under the control of a late promoter, it is expressed only at the end of the virus replication cycle, at a stage when the cellular functions are already altered, and in particular the functions which bring about the post-translational modifications (for example the glycosylation).
Some teams have sought to solve these problems by inserting the heterologous gene whose expression is desired, no longer under the control of a late promoter, but of an early promoter. The genes placed under the control of early promoters are highly expressed from the beginning of the viral infection. Some of these promoters, for example the promoters of the IE1 [GUARINO and SUMMERS, J. Virol. 61:7, p. 2091-2099 (1987)], IE0, and IEN genes of the AcNPV baculovirus (Autographa californica nuclear polyhedrosis virus) are, contrary to the polyhedrin or P10 promoters, perfectly recognized by the cell RNA polymerase II. The expression of genes placed under the control of these promoters does not therefore depend on viral infection. It has thus been proposed to use the IE1 and IEN promoters to express, very early, heterologous genes.
It has also been proposed to use the sequence encoding protein IE1, in combination with a promoter of another baculovirus early gene, such as the 39K promoter (the protein encoded by the IE1 gene is a trans-activation factor for the expression of other baculovirus early genes, in particular of the 39K gene), and optionally with enhancer sequences (hr1, hr2, hr3, hr4, hr5) [GUARINO et al., J. Virol. 60:1, p. 224-229 (1986)]for the construction of expression vectors; (Application PCT/WO 92/05265 in the name THE TEXAS A & M UNIVERSITY SYSTEM designating as inventors GUARINO and JARVIS).
The aim pursued, in both cases, was to obtain, in particular, vectors which can be integrated into the cellular genome so as to stably express a heterologous gene under the control of a baculovirus early promoter. However, tee use of such vectors poses other problems. Indeed, neither the site nor the stability of integration into the cellular genome are controllable; now, these two parameters determine to a large extent the expression of the integrated gene.
Another approach to the problems posed by the expression of a heterologous protein in an animal cell consists in attempting to construct de novo an expression vector which, on the one hand, would replicate autonomously, and on the other hand would be maintained in the cell and be transmitted to its progeny. This means that this vector should contain at least one sequence ensuring the function of a replication origin which is active in the cell, as well as sequence ensuring its maintenance in the cell, and its segregation in the daughter cells during cell division.
Sequences active as extrachromosomal replication origin have been known for a long time in yeasts: these sequences, which are present in DNA portions, are called ARS (autonomously replicating sequences). The ARS elements possess two consensus sequences: one core sequence (A domain) which is necessary for the ARS function [KEARSEY, Cell., 37, 299-307, (1984)]but which, by itself, is not sufficient to permit the autonomous replication, and requires the presence of additional flanking sequences, and a 3' consensus sequence (C domain) situated downstream of the 3' end of the strand rich in T residues of the core sequence.
Although sequences similar to the consensus sequences of the ARS elements have been described in animal cells, their possible role in the replication of DNA in these cells has never been established. Several studies have been carried out with the aim of identifying sequences which are effectively active as replication origin in animal cells, in particular in mammalian cells [KRYSAN et al., Mol. Cell. Biol. 9, p. 1026-1033, (1989); HOLST et al., Cell 52, p. 355-365, (1988); cf also review by UMEK et al., Biochim. and Biophys. Acta, 1007, p. 1-14, (1989)]. However, in most cases, these studies have not resulted in the production of sequences permitting the replication and the maintenance of extrachromosomal genetic information.
In the case of insect cells, the only replication origin which has been identified is that of the Drosophila melanogaster mitochondrial DNA [SUGINO, Biochem. Biophys. Res. Commun. 91, p. 1321-1329, (1979)]. Although ARS sequences have also been localized in the genome of drosophila [GRAGEROV et al., Nucl. Acids Res. 16, p. 1169-1180, (1988)], their possible activity as replication origin has not been shown. As regards insect viruses, the presence, in at least 2 regions of the AcNMPV baculovirus genome, of sequences which behave like ARS elements when they are introduced into yeast cells, have been described [HOOFT VAN HIDDEKINGE et al., Arch. Virol. 88, p. 279-284, (1986)]. More recently, 4 fragments containing ARSs which are functional in S. cerevisiae have been identified [LEE et al., Virus Research, 24, p. 249-264, (1992)]in the genome of another baculovirus: the Christoneura fumiferana NPV baculovirus. Analysis of the sequence of the one among these fragments which has the highest ARS activity shows a region rich in A+T associated With a region of curved DNA. Although no sequence corresponds exactly to the core consensus sequence of the yeast ARS elements, 13 regions of sequences close to that of these elements (9 or 10 base pairs out of 11) have been identified. However, no indication is given on the functionality of these sequences in eukaryotic cells other than yeast cells. In particular, they are not presumed to be involved in the viral replication, all the more so since it has recently been shown [PEARSON et al., Science, 257, p. 1382-1384, (1992)]that in the AcNPV baculovirus, replication origins of the DNA are situated in the repetitive sequences, called hr sequences, of which each comprises several imperfect palindromes which are very similar to each other. These sequences, which are present in 6 regions distributed along the AcNPV genome, were previously known for their expression-enhancing properties [cf. publication by GUARINO et al. (1986), previously cited]. Deletion analysis of the hr sequences has made it possible to demonstrate that a sequence containing a single complete palindrome permitted the replication. Other authors [KOOL et al., Virology, 192, p. 94-101, (1993)]have described the detection and functional analysis of sequences present in the DNA of defective viruses, and which can serve as replication origin for AcNPV in the infected cells. The activity linked to the viral replication could be localized essentially in the 1,000 base pair region containing the highly repetitive DNA hr5.